Non-steroid and non-prostanoid inhibitors of steroid and prostaglandin transforming enzymes

ABSTRACT

Compounds such as 1-(4&#39;-nitrophenyl)-2-propen-1-ol are disclosed which are non-steroidal mechanism-based inactivators of rat liver 3α-hydroxysteroid dehydrogenase. The corresponding ketones are time dependent inactivators of cyclooxygenase (PGH 2  -synthase).

GOVERNMENT SUPPORT

The government has rights in this invention pursuant to NIH Grant GM33464, awarded by the Department of Health and Human Services.

RELATED APPLICATIONS

This is a division of application Ser. No. 08/114,737, filed on Aug. 30,1993, which in turn is a Divisional application of Ser. No. 756,505,filed Sep. 9, 1991, U.S. Pat. No. 5,258,296, issued on Nov. 2, 1993,which in turn is a continuation-in-part of Ser. No. 539,371, filed Jun.18, 1990 U.S. Pat. No. 5,118,621 issued on Jun. 2, 1992, which is acontinuation of Ser. No. 07/187,832 filed on Apr. 29, 1988, abandoned.

FIELD OF THE INVENTION

This invention relates to non-steroidal and non-prostanoid compoundswhich function as mechanism-based inhibitors for 3α-hydroxysteroiddehydrogenase, hydroxyprostanglandin dehydrogenases and prostaglandin Fsynthase. The compounds are also irreversible inhibitors forcyclooxygenase.

BACKGROUND OF THE INVENTION

Hydroxysteroid dehydrogenases (HSD's) are a family of enzymes which playa pivotal role in the regulation of steroid hormone action. Theseenzymes catalyze the interconversion of secondary alcohols to ketones ina positional and stereospecific manner on the steroid nucleus and sidechain. They require nicotinamide dinucleotide (phosphate), NAD(P)⁺, ascofactor. For example, 3α-hydroxysteroid dehydrogenase, (EC 1.1.1.50;3α-HSD) catalyzes the reduction of 5α-dihydrotestosterone (a potentandrogenic steroid hormone) to 5α-androstan-3α,17β-diol (a weakandrogen). In this reaction, a 3-ketosteroid is converted to a3α-hydroxysteroid and, as a result, the potency of the steroid hormoneis decreased by five orders of magnitude. Other HSD's carry outreactions of similar importance in the regulation of estrogen, progestinand glucocorticoid action. As a family, HSD's represent target enzymesfor drug development.

Research groups headed by Dr. Cecil H. Robinson (Johns HopkinsUniversity) and by Dr. Douglas F. Covey (Washington University School ofMedicine) have concentrated on the development of steroidal suicidesubstrates for HSD's. [BBRC, 1981, 101;2, 495-501; Steroids, 1982, 40;1,109-119; Steroids, 1979, 34;2, 199-206; Biochemistry, 1986, 25;23,7295-7300]. Suicide substrates are one class of mechanism-basedinhibitors which mimic the normal substrate and are transformed by theenzyme's catalytic mechanism to highly reactive alkylating agents whichthen inactivate the enzyme by forming a covalent bond at the activesite. In this manner, the enzyme catalyzes its own destruction. Thesecompounds have the potential to be highly selective since they areinnocuous by themselves until they are transformed by the target enzyme.

Based on the known functions of 3α-HSD, suicide substrates for thisenzyme may have a therapeutic use in potentiating the action ofandrogens and could be used as an adjuvant in androgen replacementtherapy. Such therapy is routinely used in the treatment of hypogonadismof pituitary and testicular origin. Androgens are also essential for themaintenance of male fertility. They can be used as anabolic steroids andcan act as anti-estrogens.

3α-HSD and several other hydroxysteroid dehydrogenases have beenreported to catalyze the oxido-reduction of non-steroidal substrates.Examples include 3β-HSD from Pseudomonas testosteroni which reducescyclohexanone as well as bicyclic and tricyclic ketones [Prog. inHormone Res., 1967, 23, 349-373], 3α-HSD and 17β-HSD from rat and mouseliver, respectively, which oxidize1,2-trans-dihydroxy-3,5-cyclo-hexadiene [Biochem. J., 1984, 222;3,601-611; J. Biol. Chem., 1983, 258;12, 7252-7255], and rat liver 3α-HSDwhich reduces aromatic ketones and quinones [Biochem. J., 1984, 222;3,601-611]. These observations suggest that suicide substrates based onnon-steroids could provide an alternative rational approach to inhibitordesign.

In the present invention, monocyclic-aromatic allylic and acetylenicalcohols are shown to be highly selective inhibitors of 3α-HSD. Thenon-steroidal nature of these compounds, coupled with the fact they areless expensive and more readily synthesized, indicate that they offerdistinct advantages over their steroidal counterparts. This approach maybe generally applicable for the development of suicide substrates forother hydroxysteroid dehydrogenases.

Certain suicide substrates of this invention based onmonocyclic-aromatic allylic and acetylenic alcohols are transformed by3α-HSD to monocyclic-aromatic vinyl and acetylenic ketones. Theseketones have the unusual property of alkylating the pyridine nucleotidebinding site of 3α-HSD. Therefore monocyclic-aromatic vinyl andacetylenic ketones may represent a general class of affinity ligands forpyridine nucleotide binding sites.

A number of additional properties have been assigned to the purified3α-HSD of rat liver cytosol which suggest that suicide substrates forthis enzyme may have broader applications. First, the enzyme canfunction as a hydroxyprostaglandin dehydrogenase and will catalyze theconversion of PGF₂ α (a non-inflammatory prostaglandin) to PGE₂ (apro-inflammatory prostaglandin) [BBRC, 1987, 148;2, 646-652]. Second,deduction of the amino acid sequence of the enzyme from its cDNAindicates that 3α-HSD has close sequence homology with prostaglandin Fsynthase (PGF-synthase) [J. Biol. Chem, 1991, 266;14, 8820-8825].PGF-synthase catalyses the conversion of PGH₂ (an unstableendoperoxy-hydroxyprostaglandin) to the primary prostaglandins whichmediate symptoms of inflammation. Third, 3α-HSD is potently inhibited ina competitive fashion by all the major classes of non-steroidalanti-inflammatory drugs (NSAID's) in rank-order of their therapeuticpotency. This inhibition occurs at concentrations that are beneath thepeak plasma concentrations observed in man [PNAS, 1983, 80;14,4504-4508]. Fourth, the anti-inflammatory drug-sensitive 3α-HSD iswidely distributed in rat tissues. It is found in high levels in tissueswhich play a role in the metabolism of prostaglandins, in, for example,the lung, heart, seminal vesicle and spleen. [Biochem. Pharm., 1985,34;6, 831-835]. Collectively, these data suggest that 3α-HSD may be analternative target for anti-inflammatory drugs.

Because 3α-HSD displays these additional properties, the suicidesubstrates based on monocyclic-aromatic allylic and acetylenic alcoholsof this invention should inactivate hydroxyprostaglandin dehydrogenasesand prostaglandin F synthase in a mechanism based manner.

While the monocyclic aromatic allylic and acetylenic alcohols displayhigh selectivity for 3α-HSD, their selectivity can be further enhancedby coupling them to appropriate functionalities to imitate NSAIDs. Oncethese coupled compounds are oxidized by 3α-HSD to the correspondingketones they have the capacity to alkylate the NSAID binding site inpreference to the NAD(P)⁺ binding site of 3α-HSD.

The monocyclic-aromatic and coupled ketones of this invention can causetime dependent inactivation of prostaglandin-H synthase (EC 1.14.99.1,PGH-synthase, cyclooxygenase). Cyclooxygenase converts arachidonic acidto prostaglandins which mediate many physiological andpathophysiological processes including inflammation. Cyclooxygenase is abifunctional enzyme, catalyzing both the oxygenation of arachidonic acidto the hydroperoxy endoperoxide prostaglandin G₂ and the reduction ofprostaglandin G₂ to the hydroxy endoperoxide prostaglandin H₂.Cyclooxygenase is the target for a variety of non-steroidalanti-inflammatory drugs. For example, aspirin is Known to inhibit theoxygenase activity of cyclooxygenase by acetylating a single internalserine located within the polypeptide chain.

This invention covers monocyclic-aromatic allylic and acetylenicalcohols, appropriately coupled compounds which imitate NSAIDS, and allenzyme-generated products which mediate enzyme inactivation.

Based upon the known functions of 3α-HSD, hydroxyprostaglandindehydrogenases, and prostaglandin synthases (PGF synthase and PGHsynthase), inhibitors for these enzymes may have a number of broad uses:(1) they may potentiate androgen action and act as adjuvants in androgentherapy; (2) they may represent suicide substrates ofhydroxyprostaglandin dehydrogenases and prostaglandin F synthase; (3)they may act as time dependent inactivators of cyclooxygenase; and (4)they may represent a new class of anti-inflammatory drugs.

SUMMARY OF THE INVENTION

Tests by the inventors indicate that 1-(4'-nitrophenyl)-2-propen-1-ol(I) and 1-(4'-nitrophenyl)-2-propyn-1-ol (II) are highly selectivemechanism-based inactivators of the enzyme 3α-HSD. ##STR1## They areoxidized by the enzyme to the corresponding α,β-unsaturated ketones,1-(4'-nitrophenyl)-2-propen-1-one (III) and1-(4'-nitrophenyl)-2-propyn-1-one (IV). ##STR2## These ketonessubsequently inactivate the enzyme by a Michael addition, as shown inScheme 1 for 1-(4'-nitrophenyl)-2-propen-1-ol, wherein X=anynucleophilic amino acid residue on the enzyme, ENZ. ##STR3##

The ketones, (III) and (IV), are stoichiometric inactivators of 3α-HSDand can be used to titrate the purified enzyme. Current evidenceindicates that the pyridine nucleotide binding domain is labeled by theenzyme-generated ketones in preference to the steroid binding site.

By converting the α,β-unsaturated alcohols to analogs that imitateNSAID's, the resulting compounds have the potential to act as NSAIDbased suicide substrates of 3α-HSD. Exemplary inactivation mechanismsinvolving N-phenyl-anthranilic acids (V), 1-methyl-pyrrole acetic acids(VI) or aryl propionic acids (VII) are provided in Scheme 2. Theenzyme-generated ketones would be expected to alkylate theanti-inflammatory drug binding site 3α-HSD.

Tests by the inventors indicate that 1-(4'-nitrophenyl)-2-propen-1-one(III) and 1-(4'-nitrophenyl-2-propyn-1-one (IV) also irreversiblyinactivate prostaglandin synthases in a time dependent manner.

Based on the finding that ketones (iii) and (iv) inactivatecyclooxygenase, α,β-unsaturated alcohols that imitate NSAIDs have thepotential to act as pro-drugs. Upon oxidative metabolism to aα,β-unsaturated ketones, the activated compounds could alkylate theanti-inflammatory drug binding site of cyclooxygenase. ##STR4##

This invention therefore relates to a method of inactivating an enzymeselected from hydroxysteroid dehydrogenases, hydroxyprostaglandindehydrogenases, and prostaglandin synthases such as PGF-synthase andPGH-synthase, comprising contacting said enzyme or tissue suspected tocontain said enzyme with a compound of the formula: ##STR5## where R₁ isselected from the groups consisting of CH═CH₂, CH═CH--OMe, CH═CH--OEt,C.tbd.CH, C.tbd.C--OMe, and C.tbd.C--OEt;

R₂ is selected from the groups consisting of NO₂, Z, CH₂ Z, CHZ₂, CZ₃,COOH, NH₂ and OH;

R₃ and R₄ are independently selected from the groups consisting of H,NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ and OH;

R₅ is selected from the groups consisting of: ##STR6## and Z is ahalogen atom.

This invention further relates to a method of inactivating an enzymeselected from hydroxysteroid dehydrogenases, hydroxyprostaglandindehydrogenases, and prostaglandin synthases such as PGF-synthase,comprising contacting said enzyme or tissue suspected to contain saidenzyme with a compound of the formula: ##STR7## where R₁ is selectedfrom the groups consisting of CH═CH₂, CH═CH--OMe, CH═CH--OEt, C.tbd.CH,C.tbd.C--OMe, and C.tbd.C--OEt;

R₂ is selected from the groups consisting of NO₂, Z, CH₂ Z, CHZ₂, CZ₃,COOH, NH₂ and OH;

R₃ and R₄ are independently selected from the groups consisting of H,NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ and OH;

R₅ is selected from the groups consisting of ##STR8## and Z is a halogenatom, under conditions which allow said enzyme or another moiety tocatalyze the oxidation of said compound to the corresponding ketone.

Still further, this invention relates to novel compounds which may beused in the above-mentioned methods. These compounds have the formulas:##STR9## where A is --C(O)-- or --CH(OH)--;

R₁ is selected from the groups consisting of CH═CH₂, CH═CH--OMe,CH═CH--OEt, C.tbd.CH, C.tbd.C--OMe, and C.tbd.C--OEt;

R₂ is selected from the groups consisting of NO₂, Z, CH₂ Z, CHZ₂, CZ₃,COOH, NH₂ and OH;

R₃ and R₄ are independently selected from the groups consisting of H,NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ and OH;

R₅ is selected from the groups consisting of: ##STR10## and Z is ahalogen atom, provided that, for compounds of formula (v), when R₂ ═NO₂and R₃ and R₄ ═H, then A═--CH(OH)-- and R₁ is other than C.tbd.CH.

Still further, this invention relates to pharmaceutical compositionscomprising one of the compounds of formulas (i), (ii), (iii) and (iv)above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a HPLC chromatogram identifying the product of the enzymecatalyzed oxidation of 1-(4'-nitrophenyl)-2-propen-1-ol.

FIGS. 2a and 2b are plots showing the time dependent inactivation of3α-HSD by 1-(4'-nitrophenyl)-2-propen-1-ol.

FIGS. 3a and 3b are plots showing the time dependent inactivation of3α-HSD by 1-(4'-nitrophenyl)-2-propen-1-one.

FIGS. 4a, 4b, and 4c are plots showing the time dependent inactivationof cyclooxygenase by 1-(4'-nitrophenyl)-2-propen-1-one, by1-(4'-nitrophenyl)-2-propyn-1-one, and aspirin, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Studies show that the allylic alcohol 1-(4'-nitrophenyl)-2-propen-1-olcan be oxidized by 3α-HSD in the presence of NAD⁺ (K_(m) =2.02 mM andV_(max) =0.58 μmoles/min/mg), to yield the active Michael acceptor1-(4'-nitrophenyl)-2-propen-1-one, which has been identified by HPLC(high performance liquid chromatography), as shown in FIG. 1.

Enzymatic oxidation of the allylic alcohol (I) by 3α-HSD in the presenceof micromolar concentrations of NAD⁺ results in a time-dependent loss of3α-HSD activity, as seen in FIG. 2a. The presence of NAD⁺ is obligatoryfor the inactivation of 3α-HSD by the allylic alcohol. Analysis of thisdata by the method of Kitz and Wilson, [J. Biol. Chem., 1962, 237;10,3245-3249], yields a K_(i) of 1.2 mM for the alcohol and a t_(1/2) lifefor the enzyme at a saturating allylic alcohol concentration of 8seconds, as shown in FIG. 2b.

The enone (III), produces inactivation of 3α-HSD at a rate which is toofast to accurately measure under the conditions employed. Incubation ofsub-stoichiometric quantities of enone (III), with purified 3α-HSDresults in the titration of 3α-HSD activity, as shown in FIG. 3.

Comparison of the rate of allylic alcohol oxidation with that of enzymeinactivation suggests that 10 molecules or less of the allylic alcohol(I) are oxidized for every molecule of enzyme inactivated. This ratio,known as the partition ratio, is formally defined as k_(cat) /k_(inact); for 1-(4'-nitrophenyl)-2-propen-1-ol it is estimated to be equal to5.3, which is close to the theoretical limit of 1.

The acetylenic alcohol, 1-(4'-nitrophenyl)-2-propyn-1-ol (III), isoxidized by the enzyme with a K_(m) and V_(max) of 750 mM and 0.3μMoles/min/mg, respectively, to yield the acetylenic ketone. Onceformed, the acetylenic ketone (IV) is highly reactive. Evidence that theacetylenic ketone is the product of enzymatic oxidation was obtained bytrapping the enzymatic oxidation product as stable monothioether adductswith 2-mercaptoethanol. The adducts trapped in the enzymatic reactionwere then characterized by comparing their retention times to syntheticstandards of monothioether adducts by HPLC. Like the enone (III), theacetylenic ketone (IV) inactivates 3α-HSD in a rapid and stoichiometricmanner.

Irrespective of whether the allylic alcohol, acetylenic alcohol or theircorresponding ketones are used to inactivate 3α-HSD, inactivation isaccompanied by stable covalent bond formation between the enzyme and theinactivator. Thus, enzyme activity does not return upon either gelfiltration or extensive dialysis of the inactivated enzyme.

Three types of experimental evidence indicate that both the enone (III)and the acetylenic ketone (IV) alkylate the pyridine nucleotide bindingdomain in preference to the steroid binding site of the enzyme. First,steady state kinetic measurements indicate that the enone (III) and theacetylenic ketone (IV) are competitive inhibitors of NAD⁺ binding andnon-competitive or mixed inhibitors of androsterone binding. Second,NAD⁺ can provide complete protection of 3α-HSD from inactivation by(III) and (IV), whereas androsterone and other compounds which arecompetitive for the steroid binding site slow the rate of inactivationbut do not completely block this event. Third, chromatographicexperiments using the immobilized nucleotide analog Cibacron Blueindicate that both native enzyme and enzyme inactivated with the steroidaffinity alkylator, 17β-bromoacetoxy-5α-androstan-17β-ol-3-one, bindtightly to the immobilized nucleotide analog. By contrast, enzymeinactivated by either (III) or (IV) has no affinity for the immobilizednucleotide analogue. This experimental evidence implies that thealkylators (III) and (IV) covalently modify the nucleotide bindingdomain of 3α-HSD.

Although a number of HSD's oxido-reduce a variety of non-steroidalsubstrates, the non-steroidal suicide substrates covered by thisinvention appear to be highly selective agents for 3α-HSD. A broadscreen was conducted in which the allylic (I) and acetylenic alcohols(II) were examined as suicide substrates of the following enzymes:3α-HSD and 3(17)β-HSD (Pseudomonas testosteroni), 3α,20α-HSD(Streptomyces hydrogenase), 20α-HSD (rat ovary), 3β-HSD (rat liver),alcohol dehydrogenase (yeast), lactate dehydrogenase (porcine heart) and3α-HSD (rat liver). The results indicated that only rat liver 3α-HSDeffectively oxidized the alcohols and was subsequently inactivated bythe enzyme generated ketones.

Studies indicate that the vinyl ketone 1-(4'-nitrophenyl)-2-propen-1-one(iii) and the acetylenic ketone 1-(4'-nitrophenyl)-2-propyn-1-one (iv)cause time and concentration dependent inactivation of a semi-purifiedpreparation of cyclooxygenase obtained from sheep seminal vesiclemicrosomes. For compound (iii), the concentration required to inactivate50% of the enzyme activity was 837 μM and the t_(1/2) life at saturationwas 1-2 minutes. By contrast, acetyl salicylate at a concentration of100 μM inactivated 50% of this semi-purified preparation ofcyclooxygenase over 20 hours, as shown in FIGS. 4(a-c).

Preparation of the Compounds

Latent Michael acceptors of the general formula (iii) may be synthesizeddirectly from the corresponding benzaldehyde and vinyl or acetylenemagnesium bromide, as shown in Scheme 3. Subsequent oxidation yields theactive Michael acceptor of general formula (i).

Scheme 3

The synthesis of N-phenyl-anthranilic acid analogues incorporating thelatent aromatic Michael acceptors described above may be undertaken by anumber of routes. The two principal reactions used to synthesize##STR11## N-phenyl-anthranilic acids are the Ullmann reaction (forthorough reviews: Acheson, "Acridines" in The Chemistry of theHeterocyclic Compounds (Interscience, New York, 1956); Albert, "TheAcridines" (Edward-Arnold Co. London, 1951) anddiphenyliodonium-2-carboxylate (DPIC) coupling [J. Org. Chem., 1980,45;11, 2127-2131], both are shown schematically below. Because they arelargely complimentary in the substitutions they allow, diversefunctionalities can be incorporated into the N-phenyl-anthranilic acidnucleus, as in Scheme 4.

Scheme 4 ##STR12##

The 1-methyl-pyrrole acetic acid and arylpropionic acid analogs may beprepared by analogous methods known in the art.

The preparation of the compounds of this invention will be furtherillustrated by the following examples.

Example 1

Preparation of 1-(4'-nitrophenyl)-2-propen-1-ol, (I) and1-(4'-nitrophenyl)-2-propen-1-one, (II).

p-Nitrobenzaldehyde (1 g, 6.6 mMoles) and 30 ml of dry tetrahydrofuran(THF) was stirred at 0° C. in a septum sealed flask equipped with apressure equalizing funnel. After purging with dry nitrogen, 7 ml of 1Mvinyl magnesium bromide in THF was added dropwise under a nitrogenatmosphere. After addition of the Grignard reagent, the reaction wasallowed to stir at room temperature for 30 min. The bulk of the THF wasremoved under vacuum and the reaction was quenched by pouring it into aslurry containing 200 ml of water, crushed ice and 100 ml of ether. Thefinal pH of the slurry was adjusted to near neutral by dropwise additionof 2M sulfuric acid. The product was extracted with ether and the pooledextracts were washed twice with 30 ml of water and twice with 30 ml ofsaturated NaCl. Ether was removed under vacuum yielding a dense yellowoil which was chromatographed on preparative silica TLC plates usingtoluene:acetonitrile 10:1 (v/v) as running solvent. Elution of theproduct from silica with methanol yielded 700 mg of semi-crystalline1-(4'-nitrophenyl)-2-propen-1-ol. Recrystallization from toluene andthen ethanol gave needles of pure product. IR: (chloroform) (cm⁻¹) --OH,3600, 1040; intermolecular --OH, --C═CH₂, 3020, 2870, 985, 935 aromatic,1607, 858; --NO₂, 1510, 1350, 872. NMR: δ (p.p.m.) ([² H]chloroform),2.2 (1H, broad s, --OH, D₂ O exchangeable), 5.26 (1H, d, J₂,3 cis 10.6Hz, J₃,3 gem 1 Hz, --C═CH₂), 5.32 (1H, broad m, --CH═C), 5.42 (1H, d,J₂,3 trans 17.2 Hz, J₃,3 gem 1 Hz --C═CH₂), 6.0 (1H, m, C--CH═C), 7.4(2H, d, J 9 Hz, aromatic), 8.21 (2H, d, J 9 Hz, aromatic). UV_(max) 272nm, ε= 10,500M⁻¹ cm⁻¹ ; Mp.=55° C.

Oxidation of 1-(4'-nitrophenyl)-2-propen-1-ol (200 mg) to yield1-(4'-nitrophenyl)-2-propen-1-one was conducted in 5 ml of acetone atroom temperature by the dropwise addition of Jones reagent, (CrO₃ /H₂SO₄) with stirring. Addition of the reagent was continued until thereaction remained yellow for more than 5 min. Addition of 0.2 ml ofmethanol consumed the excess oxidant. The reaction was poured into 40 mlof water and neutralized with 1M NaOH. The product was extracted intotoluene until completion and the pooled extracts were washed withsaturated Na₂ CO₃ followed by saturated NaCl. Removal of the tolueneunder vacuum gave a semi-crystalline mass of1-(4'-nitrophenyl)-2-propen-1-one. Recrystallization from toluene thenethanol gave pure needles of product. IR: (chloroform) (cm⁻¹) C═O, 1670;C═CH₂, 1002, 911; aromatic, 1610, 855; --NO₂, 1520, 1350, 870. NMR: δ(p.p.m.) ([² H]chloroform) 6.15 (1H, m), 6.6 (1H, m), 7.2 (1H, m), 8.1(2H, d, J 8 Hz, aromatic), 8.3 (2H, d, J 8 Hz, aromatic); UV_(max) 266nm, ε₂₆₆ =17,700M⁻¹ cm.sup. -1 ; MP=89°-90° C.

Example 2

Preparation of 1-(4'-nitrophenyl)-2-propyn-1-ol, (II) and1-(4'-nitrophenyl)-2-propyn-1-one, (IV).

Synthesis of the acetylenic alcohol (II), and ketone, (IV), can beaccomplished by the general method outlined for the allylic alcohol (I),and the enone (II), by substituting acetylene magnesium bromide forvinyl magnesium bromide. Acetylenic alcohol (II) IR: (chloroform) (cm⁻¹)--OH, 3590, 1040; intermolecular --OH, 3550-3350; --C.tbd.CH, 3300,2125; aromatic 1590, 844; NO₂, 1500, 1340, 852; UV_(max) =269 nm inacetonitrile, ε₂₅₉ =6,600M⁻¹ cm⁻¹. NMR: δ (p.p.m.) ([² H] chloroform)2.75 (1H, d, J 2 Hz, --CH--C.tbd.C--), 3.0 (1H, broad s, D₂ Oexchangeable, --COH--C.tbd.C), 5.6 (1H, d, J 2 Hz, --C.tbd.CH), 7.6 (2H,d, J 9 Hz, aromatic), 8.2 (2H, d, J 9 Hz, aromatic). MP=57° C.

Acetylenic ketone (III): IR: (chloroform) (cm⁻¹) C═O, 1660; --C.tbd.CH,3300, 2100; aromatic, 1605, 855; NO₂, 1520, 1340, 870. UV_(max) =271 nmin acetonitrile, ε₂₇₁ =16,620M⁻¹ cm⁻¹. NMR: δ (p.p.m.) ([² H]chloroform) 3.58 (1H, s, --C.tbd.CH), 8.37 (4H, s, aromatic).MP=125°-127° C.

Example 3

Preparation of 2'-nitro-5'-(2-propen-1-ol)-anthranilic acid (V).

(a) As shown in Scheme 5, oxidation of 3-fluoro-4-nitrotoluene by one oftwo routes affords the corresponding aldehyde. Then, as in Example 1,addition of vinyl magnesium bromide will yield the corresponding allylicalcohol. Subsequent coupling with 2-amino-benzoic acid via an Ullmannreaction will yield the N-phenyl-anthranilic acid containing a latentMichael acceptor. ##STR13##

Synthesis of the active Michael acceptor, the last step above, can beaccomplished by a number of mild oxidants including those described byD. Swern, Synthesis, 1981, 139, 165-185, (1981), MnO₂ [Synthesis, 1976,133, 65-76], or chromic anhydride in hexamethyl phosphoric triamidewhich is specific for allylic alcohols [Synthesis, 1976, 133, 394-398].

(b) As shown in Scheme 6, oxidation of 3-bromo-4-nitrotoluene affordsthe corresponding aldehyde. Addition of vinyl magnesium bromide yieldsthe corresponding allylic alcohol. Subsequent coupling with2-amino-benzoic acid via an Ullmann reaction provides theN-phenyl-anthranilic acid containing a latent Michael acceptor.##STR14##

(c) As shown in Scheme 7, coupling of 3-bromo-4-nitrotoluene with2-amino-benzoic acid via an Ullmann reaction yields anN-phenyl-anthranilic acid. After protection of the acid with2-amino-2-methyl-1-propanol, oxidation affords the correspondingaldehyde. Addition of vinyl magnesium bromide and deprotection providesthe N-phenyl-anthranilic acid containing a latent Michael acceptor.##STR15## Figure legends

FIG. 1 shows the identification of the enzyme catalyzed oxidationproduct of the allylic alcohol (I) by HPLC. Systems (1 ml) containing1.43 μg of enzyme, 2.3 mM NAD⁺ and 1.3 mM allylic alcohol in 0.1Mpotassium phosphate buffer pH 7.0 with 4% acetonitrile as a co-solventwere incubated at 25° C. until no further change in the absorbance at340 nm was observed. Portions of the reaction mixture werechromatographed on a Waters C18 Bondapak column pre-calibrated withstandards using 45% acetonitrile in water as the mobile phase. Retentiontimes for the allylic alcohol (I) and enone (III) standards (panel a);chromatogram resulting from the incubation containing 1.43 μg of enzymeand 1.3 mM allylic alcohol, omitting NAD⁺ (panel b); chromatogramresulting from the incubation containing 2.3 mM NAD⁺ and 1.3 mM allylicalcohol, omitting enzyme (panel c); chromatogram resulting from theincubation containing the complete system, 1.43 μg of enzyme, 2.3 mMNAD⁺ and 1.3 mM allylic alcohol (panel d).

FIG. 2 shows the time dependent inactivation of 3α-HSD by theconcentrations of allylic alcohol (I) indicated, in the presence andabsence of NAD⁺. 3α-HSD (24 μM of purified enzyme) was incubated insystems (30 μl) containing 1.2 mM NAD⁺, 10 mM potassium phosphate bufferpH 7.0, 1 mM EDTA and 5% acetonitrile as a co-solvent. Enzyme activitywas followed over time by diluting an aliquot of the incubation mixtureinto a 1 ml assay containing: 2.3 mM NAD⁺ and 75 μM androsterone in 0.1Mpotassium phosphate buffer pH 7.0, with 4% acetonitrile as a co-solvent.Initial rates were determined spectrophotometrically by measuring therate of NADH formation at 340 nm. Because of the 100 fold or greaterdilution of the enzyme into the assay, the initial rate measured is anindication of the enzyme activity remaining at that time. Semi-log plotsof the amount of enzyme remaining vs. time are curvilinear (panel a).Initial rates of inactivation (k_(app's)) were estimated by takingtangents to these curves. Data was replotted as 1/k_(app) vs 1/allylicalcohol from which k_(inact) (the rate constant for enzyme inactivation)and K_(i) (the binding constant of the allylic alcohol) were determined,(panel b).

FIG. 3 shows time dependent inactivation of 3α-HSD by the enone (III).Inactivation of 3α-HSD (24 μM) was conducted in incubations (60 μl) of10 mM potassium phosphate buffer pH 7.0 containing 1 mM EDTA and 4%acetonitrile as a co-solvent. After the initial enzyme activity wasdetermined (see the legend of FIG. 2 for assay composition), theconcentration of inactivator indicated (•=0 μM, o=2.1 μM, =4.9 μM,∇=10.0 μM, =15.0 μM, []24.0 μM) was added and the sample incubated at25° C. Over time, samples were withdrawn and diluted into the standardassay to determine the amount of enzyme activity remaining. A secondaryplot of the amount of enzyme inactivated vs. the amount of inhibitoradded provided the stoichiometry of inactivation when corrected fornonspecific loss of enzyme activity (panel b). The continuous linewithout points is a theoretical line for a stoichiometry of 1:1.

FIG. 4 shows the time dependent inactivation of cyclooxygenase by thevinyl ketone 1-(4'-nitrophenyl)-2-propen-1-one (FIG. 4a), the acetylenicketone 1-(4'-nitrophenyl)-2-propyn-1-one (FIG. 4b), and aspirin (FIG.4c). Semi-purified cyclooxygenase (480 μg) was pre-incubated in 100 μlsystems containing the inhibitor concentrations indicated, along with 50mM Tris-HCl (pH=7.2), 100 μM EDTA, 1 mM phenol, 1 μM hematin, 1% Tween,and 10% DMSO. At the time intervals shown, 5 μl aliquots were removedand diluted into a 600 μl assay system containing 100 mM potassiumphosphate (pH=7.2), 1 mM phenol, and 100 μM arachidonic acid. The rateof oxygen consumption was then measured. The maximum rate of oxygenuptake observed in these assays was 1.875 μmoles of oxygen consumed perminute per milligram protein.

The data presented herein suggest that compounds of formulas (i), (ii),(iii) and (iv) could be used in pharmaceutical compositions useful asadjuvants in androgen therapy and as anti-inflammatory drugs. Thisinvention, therefore, further relates to pharmaceutical compositionscomprising one or more compounds of formulas (i), (ii), (iii) and (iv),or pharmaceutically acceptable salts thereof in combination with any ofthe conventional pharmaceutically acceptable carriers or diluents.Suitable pharmaceutical carriers are well known in the art and aredescribed, for example, in Remington's Pharmaceutical Sciences, E. W.Martin, a standard reference text in this field. Pharmaceuticallyacceptable salts include, but are not limited to, salts of hydrochloric,hydrobromic, sulfuric, benzenesulphonic, acetic, fumaric, oxalic, malicand citric acids, as well as hydroxides of potassium and sodium.

What is claimed is:
 1. A pharmaceutical composition comprising acompound of the formula: ##STR16## where A is --C(O)-- or --CH(OH)--;R₁is selected from the groups consisting of CH═CH₂, CH═CH--OMe,CH═CH--OEt, C.tbd.CH, C.tbd.C--OMe, and C.tbd.C--OEt; R₂ is selectedfrom the groups consisting of NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ andOH; R₃ and R₄ are independently selected from the groups consisting ofH, NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ and OH; R₅ is selected from thegroups consisting of: ##STR17## and Z is a halogen atom, or itspharmaceutically acceptable salt, provided that, for compounds offormula (v), when R₂ ═NO₂ and R₃ and R₄ ═H, then A═--CH(OH)-- and R₁ isother than C.tbd.CH.
 2. The pharmaceutical composition of claim 1 whereA is --C(O)--.
 3. The pharmaceutical composition of claim 1 where A is--CH(OH)--.
 4. The pharmaceutical composition of claim 1 where R₁ isCH═CH₂.
 5. The pharmaceutical composition of claim 1 where R₁ isC.tbd.CH.
 6. The pharmaceutical composition of claim 1 where R₂ is NO₂.7. The pharmaceutical composition of claim 1 where R₃ and R₄ are H. 8.The pharmaceutical composition of claim 1 where said compound is1-(4'-nitrophenyl)-2-propen-1-ol.
 9. The pharmaceutical composition ofclaim 1 where said compound is 1-(4'-nitrophenyl)-2-propyn-1-ol.
 10. Acompound of the formula: ##STR18## where A is --C(O)-- or --CH(OH)--;R₁is selected from the groups consisting of CH═CH₂, CH═CH--OMe,CH═CH--OEt, C.tbd.CH, C.tbd.C--OMe, and C.tbd.C--OEt; R₂ is selectedfrom the groups consisting of NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ andOH; R₃ and R₄ are independently selected from the groups consisting ofH, NO₂, Z, CH₂ Z, CHZ₂, CZ₃, COOH, NH₂ and OH; R₅ is selected from thegroups consisting of: ##STR19## and Z is a halogen atom, provided that,for compounds of formula (v), when R₂ ═NO₂ and R₃ and R₄ ═H, thenA═--CH(OH)-- and R₁ is other than C.tbd.CH.
 11. A compound of claim 10where A is --C(O)--.
 12. A compound of claim 10 where A is --CH(OH)--.13. The method of claim 10 where R₁ is CH═CH₂.
 14. The method of claim10 where R₁ is C.tbd.CH.
 15. The method of claim 10 where R₂ is NO₂. 16.The method of claim 10 where R₃ and R₄ are H.
 17. The method of claim 10where said compound is 1-(4'-nitrophenyl)-2-propen-1-ol.
 18. The methodof claim 10 where said compound is 1-(4'-nitrophenyl)-2-propyn-1-ol.